Currently available prostheses for the replacement of defective heart valves and other vascular structures may be classified as mechanical or bioprosthetic. Mechanical structures such as heart valves are manufactured from biocompatible metals and other materials such as Silastic.RTM., graphite, titanium, and Dacron.RTM.. Although mechanical valves have the advantage of proven durability in decades of use, they frequently are associated with a high incidence of blood clotting on or around the valve. This can lead to acute or subacute closure. For this reason, patients with implanted mechanical valves generally must remain on anticoagulants for as long as the valve remains implanted. Anticoagulants are inconvenient to take and impart a 3-5% annual risk of significant bleeding.
Bioprosthetic valves were introduced in the early 1960's and are typically derived from pig aortic valves or are manufactured from other biological materials such as bovine pericardium. Xenograft heart valves invariably are tanned in glutaraldehyde prior to implantation. A major rationale for the use of autologous or heterologous biological material for heart valves is that the profile and surface characteristics of this material are optimal for laminar, nonturbulent blood flow. The result is that intravascular clotting is less likely to occur than with mechanical valves. This concept has been proven in clinical use with the well-documented reduced thrombogenicity of current versions of glutaraldehyde-fixed bioprosthetic valves.
Unfortunately all such valves are limited by the tendency to fail, often catastrophically, beginning about 7 years after implantation. Few bioprosthetic valves remain functional after 12 years. Valve degeneration is particularly rapid in the young and during pregnancy. Replacement of a degenerating valve prosthesis is particularly hazardous in the elderly and in situations of emergency replacement. As a consequence, there are few real indications and many contraindications to the use of bioprosthetic valves.
Clearly, solving the problem of bioprosthetic valve degeneration would be highly desirable. Calcification appears to be the primary insult leading to degeneration. Efforts to address the calcification problem have included treating glutaraldehyde-fixed valves with compounds such as toluidine blue, sodium dodecyl sulfate and diphosphonate to reduce calcium nucleation. However, these efforts have been unsuccessful in vivo. Other approaches include removal of reactive glutaraldehyde moieties from the tissue by a chemical process. Still other approaches have included development of alternate fixation techniques, since evidence suggests that the fixation process itself contributes to calcification and mechanical deterioration. Finally, since nonviable cells present in transplanted tissue are sites for calcium deposition, various processes have been developed to remove cells from the valve matrix prior to implantation. Most of these processes appear to reduce calcification in animal models in short-term use, but clinical data are not yet available.
Another major disadvantage to bioprosthetic devices is the failure of such devices to be self-maintaining. Neither cadaveric allografts nor glutaraldehyde-fixed xenografts have significant populations of viable cells, and glutaraldehyde is highly cytotoxic. Since viable cells in the valve provide protection against the insudation of calcium, it is likely that any devitalized bioprosthesis will undergo calcification over time. Hence it is essential to the development of a durable prosthetic device that it support ingrowth and colonization of cells 1) from the host during the period after implantation, 2) from autologous or allogeneic sources before implantation, e.g., during a period of in vitro culture prior to implantation, or 3) from both the host after implantation and from various sources prior to implantation.
Various detergents and nucleases have been used in the past to obtain extracellular matrix from body sources for use as potential graft materials. Detergent treatment of a glutaraldehyde-fixed body structure is disclosed in U.S. Pat. No. 4,323,358 as a method for retarding mineralization after implantation. Decellularization of unfixed, untreated body structures by detergent methods is disclosed in U.S. Pat. No. 4,352,887 as a means for producing a substrate for cell culture, and in U.S. Pat. No. 4,801,299 as a means for producing sterile body implants. U.S. Pat. No. 4,776,853 discloses a specific process for achieving decellularization by a combination of nonionic and anionic detergents, deoxyribonuclease (DNAse) and ribonuclease (RNAse).
The field of tissue and organ transplantation is growing rapidly as a result of a number of advances in the areas of organ preservation, surgical techniques, and immunosuppressive agents. As a result, shortages of implantable material are often the major obstacle in the use of bioprosthetic implants. As used herein, the term "autologous" refers to cells, tissues or other biological structures derived from the same individual designated to receive the implant. The term "allogeneic" refers to cells, tissues or other biological structures taken from other members of the same species. The term "xenogeneic" refers to cells, tissues or other biological structures taken from a member of a species other than the species of the individual receiving the implant.
Shortages of implantable materials for human patients are particularly acute for heart valves, where autologous structures such as the pulmonic valve can only infrequently be used as a source of replacement material, and allogeneic implantable materials are limited. Improved methods of cryopreservation have increased the number of available allogeneic bioprosthetic implants. Extending the use of cryopreservation to xenogeneic body sources is disclosed in U.S. Pat. No. 5,336,616. That patent describes a method for processing and preserving acellular collagen-based tissue matrix for transplantation that includes a detergent-based decellularization step. Decellularization is followed by cryopreservation and dehydration. On rehydration, the tissue is inoculated with viable autologous or allogeneic cells.
A number of specific conditions that affect use of xenogeneic material for heart valve implantation have yet to be fully addressed. These include the exacting requirements for mechanical integrity in short- and long-term use, as unscheduled replacement of these structures is difficult and risky. Long-term durability is affected not only by the harvesting and decellularization processes, but by the ability of cells to enter and carry out maintenance functions in the implanted tissue. That viable cells are an essential determinant of valve survival is clear from longitudinal studies in allograft recipients. In these studies, proper allograft preservation can maximize the number of viable cells remaining in the tissue as determined by matrix protein synthesis. Preservation techniques that do not promote cell survival, such as long term storage at 4.degree. C., are associated with reduced in vivo durability and increased reoperation rates.
The repopulation of matrix by contiguous cells in the host, or by inoculated autologous or allogeneic cells in tissue culture, can be critical to development of a successful implant. In turn, the characteristics of the matrix can be critical to the repopulation ("recellularization") process. For example, failure to effectively reduce the dense cellularity of the donor aortic root is likely to be a significant obstacle to cell ingrowth, due to the physical barrier created by the cellular architecture of the root, or to the effect of contact inhibition between in-migrating host cells and the remaining in situ donor cells.
Previously described processing methods have been developed and tested with the goals of reducing antigenicity and mineralization of the xenograft, but have dealt only incidentally with the problem of recellularization. Thus, the adequacy of decellularization and the ability of the decellularized matrix to support ingrowth by autologous or allogeneic cells remains to be demonstrated. Moreover, the physical constraints imposed by the method of implantation have not been considered in envisioning how host cells may migrate into and repopulate a decellularized matrix following implantation. For example, methods of implantation may include use of a Dacron.RTM. sewing ring in the case of xenograft heart valves, or freehand attachment via the root structure in the case of allogeneic heart valves. Each of these methods presents distinctive challenges for obtaining adequate recellularization of the decellularized matrix. The methods disclosed herein provide a useful substrate for ingrowth by host cells following implantation due to enhanced removal of cells from the host/graft interface.